Method for the idenification of ligands

ABSTRACT

The present invention relates generally to a method of identifying ligands that modulate protein-protein interactions. More particularly, the present invention relates to methods of determining agonists or antagonists of a co-regulator dependent target molecule based on the ability to modify the stability of the target molecule.

This application claims priority benefit of U.S. Provisional ApplicationNo. 60/398,023 filed Jul. 24, 2002, which is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to a method of identifyingligands for protein-protein interactions whose affinity is modulated byligands or allosteric regulators. More particularly, the presentinvention relates to methods of determining agonist or antagonistligands of a receptor based on the ability to modify the stability ofthe receptor when in the presence of a co-regulator.

BACKGROUND OF THE INVENTION

A central theme in signal transduction and gene expression is theconstitutive or inducible interaction of protein-protein modulardomains. Knowledge of ligands that can potentiate these interactionswill provide information on the nature of the molecular mechanismsunderlying biological events and on the development of therapeuticapproaches for the treatment of disease. Existing methods for theidentification of ligands are cumbersome and limited particularly in thecase of proteins of unknown function.

Nuclear receptors are members of a superfamily of transcription factorscontrolling cellular functions including reproduction, growthdifferentiation, lipid and sugar homeostasis. Their function isregulated by a diverse set of ligands (xenobiotics, hormones, lipids andother known and undiscovered ligands). To date 48 nuclear receptors havebeen identified, 28 with known ligands and the remaining ones areclassified as orphans. The biology of the receptors is complex andtissue specific (Shang & Brown, Science 295:2465-2468, 2002) and themolecular mechanism of action appears to be a function of preferentialrecruitment of accessory proteins, referred as co-regulators, thatmodulate the function of these receptors in a ligand independent ordependent fashion. Recruitment of the appropriate co-regulator canresult in gene transcription or repression.

Panvera offers reagents for the discrimination of agonist fromantagonist ligands for the estrogen receptor subtype beta and haspresented publicly data on the preferential recruitment of co-activatorproteins (Bolger et al., Environmental Health Perspectives 106:1-7(1998); Panvera corporate presentation presented at the Orphan ReceptorMeeting San Diego, June 2002). Their reagents are used in assays basedon fluorescence resonance energy transfer (FRET).

There are publications on similar assays for other nuclear receptors(ER-α, the ERR and PPAR family) that are also based on FRET (Zhou etal., Molecular Endocrinology 12:1594-1604 1998)), (Coward et al.,98:8880-8884, (2001)). Similar experiments have been done using Biacoretechnology (Cheskis et al., J. Biological Chemistry 11384-11391(1997))(Wong et al.; Biochemistry 40:6756-6765 (2001)).

Cellular assays exist where the readout is gene expression (Camp et al.,Diabetes 49:539-547 (2001))(Kraichely et al., Endocrinology,141:3534-3545, (2000)). For example, Karo-Bio has developed a geneexpression readout assay to include conformational sensitive peptideprobes for discrimination of agonist from antagonist ligands for nuclearhormone receptors (Paige et al., PNAS 96:3999-4004 (1999)),(presentation by Karo-Bio at the Orphan Receptor Meeting, San Diego June2002).

Greenfield et al., Biochemistry 40:6446-6652 (2001) reports the thermalstablization of the ER-α receptor in the presence of estradiol. However,the reference does not teach the identification of a molecule as anagonist or an antagonist of the ER-α receptor.

The art discussed above suffers from several drawbacks. For example, inthe analysis of nuclear receptors, gene expression readout assays andcell based assays, counter-screens are required to validate that ligandsor co-regulators identified interact directly with the receptor ofinterest and not through other proteins that can produce a signaltransduction or gene activation/repression assay readout. In addition,cell readout technology lacks the sensitivity in identifying weakligands (typically compounds of affinities of greater than 1 μM arerarely identified), and is only applicable to compounds that have a goodcell permeability profile. Other commercial in vitro assays require theknowledge of ligands for establishing competitive displacement assays,or the use of them as tools to validate FRET based co-regulator assays.

Thus, there is a need for an accurate, reliable technology thatfacilitates the rapid, high-throughput identification of ligands forco-regulator-dependent receptors and further identification of theireffect on the receptor when in the presence of a co-regulator.

SUMMARY OF THE INVENTION

The present invention meets such needs. The present invention provides amethod of identifying an agonist or an antagonist of aco-regulator-dependent target molecule. The method comprises providing aset of molecules that modify the stability of the target molecule andscreening one or more molecules of the set for their ability to furthermodify the stability of the target molecule in the presence of one ormore co-regulators. A further modification of the stability of thetarget molecule in the presence of a molecule of the set and aco-regulator indicates whether the molecule is an agonist or anantagonist of the target molecule when in the presence of theco-regulator.

The invention provides another method of identifying an agonist or anantagonist of a co-regulator-dependent target molecule. The methodcomprises providing a set of molecules that shift the thermal unfoldingcurve of the target molecule and screening one or more of the moleculesof the set for their ability to further shift the thermal unfoldingcurve of the target molecule in the presence of one or moreco-regulators. A further shift in the thermal unfolding curve of thetarget molecule in the presence of a molecule of the set and aco-regulator indicates whether the molecule is an agonist or anantagonist of the target molecule when in the presence of theco-regulator.

The present invention also provides a method of identifying anantagonist of a co-regulator-dependent target molecule. The methodcomprises providing a set of molecules that modify the stability of thetarget molecule and screening one or more molecules of the set for theirability to further modify the stability of the target molecule in thepresence of one or more co-activators. If there is no furthermodification of the stability of the target molecule in the presence ofa molecule of the set and a co-activator, this is an indication that themolecule of the set is an antagonist of the target molecule when in thepresence of the co-activator.

The present invention provides another method of identifying anantagonist of a co-regulator-dependent target molecule. The methodcomprises providing a set of molecules that shift the thermal unfoldingcurve of the target molecule and screening one or more of the moleculesof the set for their ability to further shift the thermal unfoldingcurve of the target molecule in the presence of one or moreco-activators. If there is no further shift in the thermal unfoldingcurve in the presence of a molecule of the set and a co-activator, thisis an indication that the molecule of the set is an antagonist of thetarget molecule when in the presence of the co-activator.

The present invention also provides a method of identifying an agonistof a co-regulator-dependent target molecule. The method comprisesproviding a set of molecules that modify the stability of the targetmolecule and screening one or more molecules of the set for theirability to further modify the stability of the target molecule in thepresence of one or more co-repressors. If there is no furthermodification of the stability of the target molecule in the presence ofa molecule of the set and a co-repressor, this is an indication that themolecule of the set is an agonist of the target molecule when in thepresence of the co-repressor.

The present invention provides another method of identifying an agonistof a co-regulator-dependent target molecule. The method comprisesproviding a set of molecules that shift the thermal unfolding curve ofthe target molecule and screening one or more of the molecules of theset for their ability to further shift the thermal unfolding curve ofthe target molecule in the presence of one or more co-repressors. Ifthere is no further shift in the thermal unfolding curve in the presenceof a molecule of the set and a co-repressor, this is an indication thatthe molecule of the set is an agonist of the target molecule when in thepresence of the co-repressor.

The present invention also provides a method for determining an agonistor an antagonist of a target molecule having an unknown function. Themethod comprises providing a set of molecules that modify the stabilityof a target molecule having an unknown function, wherein the set ofmolecules modify the stability of receptors which share biologicalfunction, and screening one or more molecules of the set for theirability to further modify the stability of the target molecule in thepresence of one or more co-regulators. A further modification of thestability of the target molecule in the presence of a molecule of theset and a co-regulator indicates whether the molecule is an agonist oran antagonist of the target molecule when in the presence of theco-regulator.

The present invention provides another method for determining an agonistor an antagonist of a target molecule having an unknown function. Themethod comprises providing a set of molecules that shift the thermalunfolding curve of a target molecule having an unknown function, whereinthe set of molecules shift the thermal unfolding curve of receptorswhich share biological function, and screening one or more molecules ofthe set for their ability to further shift the thermal unfolding curveof the target molecule in the presence of one or more co-regulators. Afurther shift in the thermal unfolding curve of the target molecule inthe presence of a molecule of the set and a co-regulator indicateswhether the molecule is an agonist or an antagonist of the targetmolecule when in the presence of the co-regulator.

An advantage of the methods of the present invention is that neithergene expression readout and cell based assays, nor the use of knownligands to establish the assay are required. The ability to generateinformation in such a direct fashion allows the discovery of drugs withdesired properties, to test therapeutic hypotheses and decrypt orphanreceptors.

By use of isolated and/or purified proteins and peptides in a singleunifying assay, one can identify ligands that are involved in modulatingprotein-protein interactions and predict biological response. Not onlycan ligands be identified, but also the intrinsic affinity for thetarget protein can be calculated which then can be used to correlate tobiological activity. The information generated can also be used toidentify ligands for orphan receptors that in turn can be used as toolsto deconvolute the biology of these proteins to test therapeutichypotheses.

Data generated by methods of the present invention does not requirecounter-screening, as changes in the melting temperature of a targetmolecule, such as a protein is a direct consequence of the thermodynamiclinkage of the binding energy of macromolecules and ligands to theprotein of interest. Further, affinities of a ligand to a targetmolecule are more sensitive (affinities of pM to mM are determined).Further, the present invention is not limited by compounds with poorcell permeability. Also, as mentioned above, the present invention doesnot require known ligands to establish an assay, making it extremelypowerful for deconvoluting orphan receptors.

Further features and advantages of the present invention are describedin detail below with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates experimental results expected for the identificationof an agonist ligand in the presence of a co-activator.

FIG. 2 illustrates experimental results expected for the identificationof an antagonist ligand in the presence of a co-activator.

DETAILED DESCRIPTION

In the following description, reference will be made to various termsand methodologies known to those of skill in the biochemical andpharmacological arts. Publications and other materials setting forthsuch known terms and methodologies are incorporated herein by referencein their entireties as though set forth in full.

In embodiments of the present invention, methods are provided for theidentification of agonists and antagonists for co-regulator-dependenttarget molecules, which are capable of unfolding, based upon moleculesthat modify the stability of the target molecule. Molecules that modifythe stability of the target molecule can be screened in the presence ofthe target molecule and one or more co-regulators for their ability tofurther modify the stability of the target molecule. Whether thestability of the target molecule is further modified is an indication asto whether the molecule is an agonist or an antagonist of the targetmolecule when in the presence of the co-regulator.

In other embodiments of the invention, methods are provided for theidentification of agonists and antagonists for co-regulator-dependenttarget molecules which involve the unfolding of a target molecule due toa thermal change. Molecules that shift the thermal unfolding curve ofthe target molecule can be screened in the presence of the targetmolecule and one or more co-regulators for their ability to furthershift the thermal unfolding curve of the target molecule. Whether thethermal unfolding curve of the target molecule is further shifted is anindication as to whether the molecule is an agonist or an antagonist ofthe target molecule when in the presence of the co-regulator.

The term “target molecule” encompasses peptides, proteins, nucleicacids, and other receptors. The term encompasses both enzymes andproteins which are not enzymes. The term encompasses monomeric andmultimeric proteins. Multimeric proteins may be homomeric orheteromeric. The term encompasses nucleic acids comprising at least twonucleotides, such as oligonucleotides. Nucleic acids can besingle-stranded, double-stranded or triple-stranded. The termencompasses a nucleic acid which is a synthetic oligonucleotide, aportion of a recombinant DNA molecule, or a portion of chromosomal DNA.

The term “target molecule” also encompasses portions of peptides,proteins, and other receptors which are capable of acquiring secondary,tertiary, or quaternary structure through folding, coiling or twisting.

The target molecule may be substituted with substituents including, butnot limited to, cofactors, coenzymes, prosthetic groups, lipids,oligosaccharides, or phosphate groups. The term “target molecule” and“receptor” are synonymous.

More specifically, the target molecules utilized in the presentinvention are co-regulator-dependent. By “co-regulator-dependent” it ismeant that the target molecule is capable of binding at least one ligandand binding at least one co-regulator. Further, the activity of thetarget molecule, whether in a ligand dependent or independent function,is dependent upon, at least in part, by a co-regulator. Co-regulatordependent target molecules include, but are not limited to, nuclearreceptors.

Nuclear receptors, and the role of co-regulators relating thereto, aredescribed in Aranda and Pascual, Physiological Reviews 81:1269-1304(2001); Collingwood et al., Journal of Molecular Endocrinology23:255-275 (1999); Robyr et al., Molecular Endocrinology 23:329-347(2000); and Lee et al., Cellular and Molecular Life Sciences 58:289-297(2001), the references incorporated by reference herein by theirentireties.

Further, the co-regulator dependent target molecules encompassvertebrate species, including, but not limited to humans, as well asinvertebrates, including but not limited to insects.

Illustratively, insects contain hundreds of nuclear receptors, for whichligands can be identified as agonists or antagonists. See Laudet, J.Molecular Endocrinology 19:207-226 (1997) and Maglich et al., GenomeBiology 2:1-7 (2001) for a discussion of nuclear receptors present invertebrates, nematodes and arthropods, the references incorporated byreference herein by their entireties.

The term “protein” encompasses full length or polypeptide fragments. Theterm “peptide” refers to protein fragments, synthetic or those derivedfrom peptide libraries. As used herein, the terms “protein” and“polypeptide” are synonymous.

The term “co-regulator” refers to chemical compounds of any structure,including, but not limited to nucleic acids, such as DNA and RNA, andpeptides that modulate the target molecule in a ligand dependent orindependent fashion. The term refers to natural, synthetic and virtualmolecules. More specifically, the term refers to a peptide orpolypeptide/protein, natural or synthetic that modulates the targetmolecule in a ligand dependent or independent fashion. The termencompasses peptides that are derived from natural sequences or fromphage display libraries. The peptide can be fragments of nativeproteins. More specifically, the term refers to co-activators andco-repressors.

The term “co-activator” refers to a molecule which binds to a targetmolecule and causes an activation of or an increase in an activity ofthe target molecule. In embodiments of the invention, the term refers tomolecules that bind to a target molecule to induce gene transcription orto induce a signaling function (e.g. signal transduction).

The term “co-repressor” refers to a molecule which binds to a targetmolecule and causes a deactivation or a decrease in an activity of thetarget molecule. In embodiments of the invention, the term refers tomolecules that bind to a target molecule to repress gene transcriptionor to repress a signaling function (e.g. signal transduction).

The term “agonist” refers to a molecule which binds to a target moleculeand induces or recruits a co-activator for binding to the targetmolecule.

In embodiments of the invention, the term “agonist” refers to a moleculethat binds to a nuclear receptor and recruits a co-activator. In theseembodiments, the term more specifically refers to a molecule that altersgene expression by inducing conformational changes in a nuclear receptorthat promote direct interactions with co-activators.

The term “antagonist” refers to a molecule which binds to a targetmolecule and induces or recruits a co-repressor for binding to thetarget molecule.

In embodiments of the invention, the term “antagonist” refers to amolecule that binds to a nuclear receptor and recruits a co-repressor.In these embodiments, the term more specifically refers to a moleculethat alters gene expression by inducing conformational changes in anuclear receptor that promote direct interactions with co-repressors.

The term “molecule” refers to a compound which is tested for binding tothe target molecule in the presence of or absence of additionalcompounds, such as co-regulators. This term encompasses chemicalcompounds of any structure, including, but not limited to nucleic acids,such as DNA and RNA, and peptides. The term refers to natural, syntheticand virtual molecules. The term includes compounds in a compound or acombinatorial library. The terms “molecule” and “ligand” are synonymous.

The terms “multiplicity of molecules,” “multiplicity of compounds,” or“multiplicity of containers” refer to at least two molecules, compounds,or containers.

The term “function” refers to the biological function of a targetmolecule, such as, e.g., a protein, peptide or polypeptide.

A “thermal unfolding curve” is a plot of the physical change associatedwith the unfolding of a protein or a nucleic acid as a function oftemperature.

The terms “bind” and “binding” refer to an interaction between two ormore molecules. More specifically, the terms refer to an interaction,such as noncovalent bonding, between a ligand and a target molecule, ora co-regulator and a target molecule, or a ligand, target molecule, anda co-regulator.

The term “modification of stability” refers to the change in the amountof pressure, the amount of heat, the concentration of detergent, or theconcentration of denaturant that is required to cause a given degree ofphysical change in a target protein that is bound by one or moreligands, relative to the amount of pressure, the amount of heat, theconcentration of detergent, or the concentration of denaturant that isrequired to cause the same degree of physical change in the targetprotein in the absence of any ligand. Modification of stability can beexhibited as an increase or a decrease in stability. Modification of thestability of a target molecule by a ligand indicates that the ligandbinds to the target molecule.

The term “further modification of stability” refers to an additionalmodification of stability of the target molecule when in the presence ofa molecule known to modify the stability of the target molecule and oneor more additional molecules. More specifically, the one or moreadditional molecules can be co-regulators.

The term “unfolding” refers to the loss of structure, such ascrystalline ordering of amino acid side-chains, secondary, tertiary, orquaternary protein structure. A target molecule, such as a protein, canbe caused to unfold by treatment with a denaturing agent (such as urea,guanidinium hydrochloride, or guanidinium thiosuccicinate), a detergent,by treating the target molecule with pressure, by heating the targetmolecule, or by any other suitable change.

The term “physical change” encompasses the release of energy in the formof light or heat, the absorption of energy in the form or light or heat,changes in turbidity and changes in the polar properties of light.Specifically, the term refers to fluorescent emission, fluorescentenergy transfer, absorption of ultraviolet or visible light, changes inthe polarization properties of light, changes in the polarizationproperties of fluorescent emission, changes in the rate of change offluorescence over time (i.e., fluorescence lifetime), changes influorescence anisotropy, changes in fluorescence resonance energytransfer, changes in turbidity, and changes in enzyme activity.Preferably, the term refers to fluorescence, and more preferably tofluorescence emission. Fluorescence emission can be intrinsic to aprotein or can be due to a fluorescence reporter molecule. The use offluorescence techniques to monitor protein unfolding is well known tothose of ordinary skill in the art. For example, see Eftink, M. R.,Biophysical J. 66: 482-501 (1994).

An “unfolding curve” is a plot of the physical change associated withthe unfolding of a protein as a function of parameters such astemperature, denaturant concentration, and pressure.

The term “modification of thermal stability” refers to the change in theamount of thermal energy that is required to cause a given degree ofphysical change in a target protein that is bound by one or moreligands, relative to the amount of thermal energy that is required tocause the same degree of physical change in the target protein in theabsence of any ligand. Modification of thermal stability can beexhibited as an increase or a decrease in thermal stability.Modification of the thermal stability of a target molecule by a ligandindicates that the ligand binds to the protein.

The term “shift in the thermal unfolding curve” refers to a shift in thethermal unfolding curve for a target molecule that is bound to a ligand,relative to the thermal unfolding curve of the protein in the absence ofthe ligand.

The term “further shift in the thermal unfolding curve” refers to anadditional shift of the thermal unfolding curve of the target moleculewhen in the presence of a molecule known to shift the thermal unfoldingcurve of the target molecule and one or more additional molecules. Morespecifically, the one or more additional molecules can be co-regulators.

The term “contacting a target molecule” refers broadly to placing thetarget protein in solution with the molecule to be screened for binding.Less broadly, contacting refers to the turning, swirling, shaking orvibrating of a solution of the target molecule and the molecule to bescreened for binding. More specifically, contacting refers to the mixingof the target molecule with the molecule to be tested for binding.Mixing can be accomplished, for example, by repeated uptake anddischarge through a pipette tip. Preferably, contacting refers to theequilibration of binding between the target protein and the molecule tobe tested for binding. Contacting can occur in the container or beforethe target molecule and the molecule to be screened are placed in thecontainer.

The term “container” refers to any vessel or chamber in which thereceptor and molecule to be tested for binding can be placed. The term“container” encompasses reaction tubes (e.g., test tubes, microtubes,vials, cuvettes, etc.). In embodiments of the invention, the term“container” refers to a well in a multiwell microplate or microtiterplate.

In embodiments of the invention, molecules that bind to the targetmolecule can be screened for their ability to bind to a target moleculein the presence of one or more co-regulators. The term “screening”refers generally to the testing of molecules or compounds for theirability to bind to a target molecule which is capable of denaturing orunfolding. The screening process can be a repetitive, or iterative,process, in which molecules are tested for binding to a protein in anunfolding assay.

As mentioned above, in accordance with embodiments of the invention,agonists or antagonists of a target molecule can be identified basedupon modification of stability of the target molecule. Molecules thatmodify the stability of the target molecule can be screened for theirability to further modify the stability of the target molecule in thepresence of one or more co-regulators.

In an embodiment, to perform the screening, one or molecules (e.g. of aset) that modify the stability of the target molecule can be contactedwith the target molecule and one of more co-regulators in each of amultiplicity of containers. The target molecule in each of thecontainers can then be treated to cause the target protein to unfold. Aphysical change associated with the unfolding of the target molecule canbe measured. An unfolding curve for the target molecule for each ofcontainers can then be generated. Each of the unfolding curves may becompared to (1) each of the other unfolding curves and/or to (2) theunfolding curve for the target molecule in the absence of (i) any of themolecules from the set and/or (ii) the co-regulators.

Based upon the generated data, one can determine whether the screenedmolecules further modify the stability of the target molecule in thepresence of the co-regulators, and thus identify whether the moleculesare agonists or antagonists of the target molecule when the presence ofthe co-regulators. A further modification of stability of the targetmolecule is indicated by a further change in the unfolding curve of thetarget molecule.

In other embodiments of the invention, an agonist or antagonist of aco-regulator-dependent target molecule can be identified by an analysisof molecules that modify the thermal stability, and more particularly,shift the thermal unfolding curve of the target molecule. Molecules thatshift the thermal unfolding curve of a target molecule can be screenedfor their ability to further shift the thermal unfolding curve of thetarget molecule in the presence of one or more co-regulators.

In an embodiment of the invention, the screening can be accomplished bycontacting the target molecule with one or more of molecules (e.g., of aset) that shift the thermal unfolding curve of the target molecule withone or more co-regulators in each of a multiplicity of containers. Themultiplicity of containers can be heated, and a physical changeassociated with the thermal unfolding curve for the target molecule as afunction of temperature can be measured for each of the containers. Athermal unfolding curve for the target molecule as a function oftemperature can then be generated. The thermal unfolding curves that aregenerated can be compared with (1) each of the other thermal unfoldingcurves and/or to (2) the thermal unfolding curve for the target moleculein the absence of (i) any of the molecules from the set and/or (ii) theco-regulators.

In embodiments of the screening method, the containers can be heated inintervals, over a range of temperatures. The multiplicity of containersmay be heated simultaneously. A physical change associated with thethermal unfolding of the target molecule can be measured after eachheating interval. In an alternate embodiment of this method, thecontainers can be heated in a continuous fashion.

In embodiments of the invention, in generating an unfolding curve forthe target molecule, a thermal unfolding curve can be plotted as afunction of temperature for the target molecule in each of thecontainers.

In an embodiment of the invention, comparing the thermal unfoldingcurves can be accomplished by comparing the midpoint temperatures, T_(m)of each unfolding curve. The “midpoint temperature, T_(m)” is thetemperature midpoint of a thermal unfolding curve. The T_(m) can bereadily determined using methods well known to those skilled in the art.See, for example, Weber, P. C. et al., 3. Am. Chem. Soc. 116:2717-2724(1994); and Clegg, R. M. et al., Proc. Natl. Acad. Sci. U.S.A.90:2994-2998 (1993).

For example, the T_(m) of each thermal unfolding curve can be identifiedand compared to the T_(m) obtained for (1) the other thermal unfoldingcurves and/or to (2) the thermal unfolding curve for the target moleculein the absence of (i) any of the molecules from the set and/or (ii) theco-regulators in the containers.

Alternatively or additionally, an entire thermal unfolding curve can besimilarly compared to other entire thermal unfolding curves usingcomputer analytical tools. For example, each entire thermal unfoldingcurve can be compared to (1) the other thermal unfolding curves and/orto (2) the thermal unfolding curve for the target molecule in theabsence of (i) any of the molecules from the set and/or (ii) theco-regulators in the containers.

Based upon the generated data, one can determine whether any of thescreened molecules further shift the thermal unfolding curve of thetarget molecule in the presence of a co-regulator, and thus identifywhether a molecule is an agonist or antagonist of the target moleculewhen in the presence of a co-regulator.

The methods of the present invention that involve determining whethermolecules that shift and/or further shift the thermal unfolding curve ofa target molecule are distinct from methods that do not involvedetermining whether molecules shift and/or further shift the thermalunfolding curve of a target molecule, such as assays of susceptibilityto proteolysis, surface binding by protein, antibody binding by protein,molecular chaperone binding of protein, differential binding toimmobilized ligand, and protein aggregation. Such assays are well-knownto those of ordinary skill in the art. For example, see U.S. Pat. No.5,585,277; and U.S. Pat. No. 5,679,582. These approaches disclosed inU.S. Pat. Nos. 5,585,277 and 5,679,582 involve comparing the extent offolding and/or unfolding of the protein in the presence and in theabsence of a molecule being tested for binding. These approaches do notinvolve a determination of whether any of the molecules that bind to thetarget molecule shift the thermal unfolding curve of the targetmolecule.

As discussed above, molecules that modify the stability of the targetmolecule can be screened for the ability to further modify the stabilityof the target molecule in the presence of a co-regulator. For example,molecules that are known to modify the stability of the target moleculescan be screened against a panel of identified co-regulators for thetarget molecule, including co-activators and/or co-repressors. Forconvenience, the molecules known to modify the stability of the targetmolecule are referred to as a “sef” of molecules.

If the stability of the target molecule is further modified in thepresence of a molecule from the set and a co-activator of the targetmolecule as compared to the target molecule and the molecule from theset alone, then this is an indication that the molecule from the set isan agonist of the target molecule when in the presence of theco-activator.

If the stability of the target molecule is further modified in thepresence of a molecule from the set and a co-repressor of the targetmolecule as compared to the target molecule and the molecule from theset alone, then this is an indication that the molecule from the set isan antagonist of the target molecule when in the presence of theco-repressor.

Similarly, molecules that shift the thermal unfolding curve of thetarget molecule can be screened for the ability to further shift thethermal unfolding curve of the target molecule in the presence of aco-regulator. For example, molecules that are known to shift the thermalunfolding curve of the target molecule can be screened against a panelof identified co-regulators for the target molecule, includingco-activators and/or co-repressors. For convenience, the molecules thatare known to shift the thermal unfolding curve of the target moleculeare referred to as a “set” of molecules.

If the thermal unfolding curve of the target molecule is further shiftedin the presence of a molecule from the set and a co-activator of thetarget molecule as compared to the target molecule and the molecule fromthe set alone, then this is an indication that the molecule from the setis an agonist of the target molecule when in the presence of theco-activator.

If the thermal unfolding curve of the target molecule is further shiftedin the presence of a molecule from the set and a co-repressor of thetarget molecule as compared to the target molecule and the molecule fromthe set alone, then this is an indication that the molecule from the setis an antagonist of the target molecule when in the presence of theco-repressor.

The present invention also provides methods for identifying agonists orantagonists of a co-regulator-dependent target molecule based on thelack of further modification of stability and/or a lack of further shiftin the unfolding curve of a target molecule.

By “lack of further modification of stability of the target molecule” or“no further modification of stability of the target molecule,” it ismeant that there is either an insignificant further change or no furtherchange in the stability of the target molecule in the presence of both amolecule from the set and a co-regulator (as compared to the targetmolecule and the molecule from the set).

By “lack of further shift in the thermal unfolding curve of the targetmolecule” or “no further shift in the thermal unfolding curve of thetarget molecule,” it is meant that there is either an insignificantfurther change or no further change in the shift of the thermalunfolding curve of the target molecule in the presence of a moleculefrom the set and of a co-regulator (as compared to the target moleculeand the molecule from the set).

In embodiments of the invention, an antagonist of aco-regulator-dependent target molecule can be identified based on thelack of further modification of stability and/or lack of further shiftin the thermal unfolding curve of a target molecule when in the presenceof a co-activator. In other embodiments of the invention, an agonist ofa co-regulator-dependent target molecule can be identified based on thelack of further modification of stability and/or lack of further shiftin the thermal unfolding curve of a target molecule when in the presenceof a co-repressor.

An antagonist of a co-regulator-dependent target molecule can beidentified by screening one or more of a set of molecules that modifythe stability of the target molecule for their ability to further modifythe stability of the target molecule in the presence of one or moreco-activators. Methods for screening the molecules from the set fortheir effect on further modifying the stability of the target moleculeare described above. If there is no further modification of thestability of the target molecule in the presence of a molecule of theset and a co-activator, then this is an indication that such molecule ofthe set is an antagonist of the target molecule when in the presence ofthe co-activator.

An antagonist can also be identified by screening one or more of a setof molecules that shift the thermal unfolding curve of the targetmolecule for their ability to further shift the thermal unfolding curveof the target molecule in the presence of one or more co-activators.Methods for screening one or more molecules of the set for their abilityto further shift the thermal unfolding curve of the the target moleculeare described above. If there is no further shift in the thermalunfolding curve of the target molecule in the presence of a molecule ofthe set and a co-activator, then this is an indication that suchmolecule of the set is an antagonist of the target molecule when in thepresence of the co-activator.

An agonist of a co-regulator-dependent target molecule can be identifiedby screening one or more of a set of molecules that modify the stabilityof the target molecule for their ability to further modify the stabilityof the target molecule in the presence of one or more co-repressors.Methods for screening the molecules from the set for their effect onfurther modifying the stability of the target molecule are describedabove. If there is no further modification of the stability of thetarget molecule in the presence of a molecule of the set and aco-repressor, then this is an indication that such molecule of the setis an agonist of the target molecule when in the presence of theco-repressor.

An agonist can also be identified by screening one or more of a set ofmolecules that shift the thermal unfolding curve of the target moleculefor their ability to further shift the thermal unfolding curve of thetarget molecule in the presence of one or more co-repressors. Methodsfor screening one or more molecules of the set for their ability tofurther shift the thermal unfolding curve of the the target molecule aredescribed above. If there is no further shift in the thermal unfoldingcurve of the target molecule in the presence of a molecule of the setand a co-repressor, then this is an indication that such molecule of theset is an agonist of the target molecule when in the presence of theco-repressor.

Methods have been described above for the identification of agonists andantagonists of a co-regulator-dependent target molecule based onproviding molecules that are known to modify the stability and/or shiftthe thermal unfolding curve of the target molecule and screening suchmolecules for their ability to further modify the stability of and/orshift the thermal unfolding curve of the target molecule. The inventionalso encompasses methods for the providing of such molecules inconjunction with the identification of such molecules as agonists orantagonists of the target molecule when in the presence of aco-regulator. Such methods are particularly useful in identifyingligands for orphan receptors, for which ligands that bind to thereceptor are not known.

Molecules that modify the stability and/or shift the thermal unfoldingcurve of the target molecule (referred to above as a “set” forconvenience) can be obtained by the screening of a multiplicity ofdifferent molecules. For example, molecules that modify the stability ofthe target molecule can be obtained by the screening of one or more of amultiplicity of different molecules for their ability to modify thestability of the target molecule. Similarly, molecules that shift thethermal unfolding curve of the target molecule can be obtained by thescreening of one or more of a multiplicity of different molecules fortheir ability to shift the thermal unfolding curve of the targetmolecule. In embodiments of the invention, the number of molecules thatcan be screened range from about one thousand to one million.

Molecules can be screened for their ability to modify the stability ofthe target molecule by a method similar to the screening methoddescribed above for identifying agonists or antagonists. For example,the target molecule can be contacted with one or more of a multiplicityof different molecules in each of a multiplicity of containers. Thetarget molecule in each of the multiplicity of containers can be treatedto cause it to unfold. A physical change associated with the unfoldingof the target molecule can be measured. An unfolding curve for thetarget molecule for each of the containers can be generated. Each ofthese unfolding curves can be compared to (1) each of the otherunfolding curves and/or to (2) the unfolding curve for the targetmolecule in the absence of any of the multiplicity of differentmolecules.

Based upon the generated data, one can determine whether any of thescreened molecules modify the stability of the target molecule. Amodification of stability of the target molecule is indicated by achange in the unfolding curve of the target molecule. If a moleculemodifies the stability of the target molecule, it can then be screenedto identify whether it is an agonist or an antagonist of the targetmolecule when in the presence of a co-regulator by the methods describedabove.

Similarly, molecules can be screened for their ability to shift thethermal unfolding curve of the target molecule by a method similar tothe screening method for identifying agonists or antagonists. Forexample, the target molecule can be contacted with one or more of amultiplicity of different molecules in each of a multiplicity ofcontainers. The containers can be heated, and a physical changeassociated with the thermal unfolding of the target molecule can bemeasured in each of the containers. A thermal unfolding curve for thetarget molecule can be generated as a function of temperature for eachof the containers.

The thermal unfolding curves can be compared with (1) each of the otherthermal unfolding curves and/or to (2) the thermal unfolding curves forthe target molecule in the absence of any of the multiplicity ofdifferent molecules. In embodiments of the invention, the T_(m) of eachthermal unfolding curve can be identified and compared to the T_(m)obtained for (1) the other thermal unfolding curves and/or to (2) thethermal unfolding curve for the target molecule in the absence of any ofthe multiplicity of molecules. Alternatively, each entire thermalunfolding curve can be compared to (1) the other thermal unfoldingcurves and/or to (2) the thermal unfolding curve for the target moleculein the absence of any of the multiplicity of different molecules.

Based upon the generated data, one can determine whether any of thescreened molecules shift the thermal unfolding curve of the targetmolecule. If a molecule shifts the thermal unfolding curve of the targetmolecule, it can then be screened to identify whether it is an agonistor an antagonist of the target molecule when in the presence of aco-regulator by the methods described above.

As discussed above, the methods of the present invention areparticularly useful in identifying ligands for orphan receptors, forwhich ligands that bind to the receptor are not known. Similarly, theinvention provides for a methods for identifying agonists andantagonists of a target molecule having an unknown function.

In an embodiment of the invention, a set of molecules is provided thatmodify the stability of a target molecule having an unknown function.This set of molecules modifies the stability of receptors which sharebiological function. The set of molecules that modify the stability ofthe target molecule can be provided by screening one or more panels ofmolecules which modify the stability of receptors which share biologicalfunction for their ability to modify the stability of the targetmolecule. Methods for providing such a set of molecules are described inmore detail in U.S. Patent Publication No. U.S. 2001/0003648, hereinincorporated by reference in its entirety.

One or more molecules of the set can be screened for their ability tofurther modify the stability of the target molecule in the presence ofone or more co-regulators. As discussed in detail above, a furthermodification of the stability of the target molecule in the presence ofa molecule of the set and a co-regulator indicates whether the moleculeis an agonist or an antagonist of the target molecule when in thepresence of the co-regulator. Embodiments of the invention include anidentification of agonists and antagonists based upon no furthermodification of stability of the target molecule.

In another embodiment of the invention, a set of molecules are providedthat shift the thermal unfolding curve of a target molecule having anunknown function. This set of molecules shifts the thermal unfoldingcurve of receptors which share biological function. The set of moleculesthat shift the thermal unfolding curve of the target molecule can beprovided by screening one or more panels of molecules which shift thethermal unfolding curve of receptors which share biological function fortheir ability to modify the stability of the target molecule. Methodsfor providing such a set of molecules are also described in more detailin U.S. Patent Publication No. U.S. 2001/0003648.

One or more molecules of the set can be screened for their ability tofurther shift the thermal unfolding curve of the target molecule in thepresence of one or more co-regulators. As discussed in detail above, afurther shift in the thermal unfolding curve of the target molecule inthe presence of a molecule of the set and a co-regulator indicateswhether the molecule is an agonist or an antagonist of the targetmolecule when in the presence of the co-regulator. Embodiments of theinvention also include an identification of agonists and antagonistsbased upon no further shift in the thermal unfolding curve of the targetmolecule.

In embodiments of the invention, a microplate thermal shift assay is aparticularly useful means for identifying ligands and identifying suchligands as agonists or antagonists of co-regulator-dependent targetmolecules. The microplate thermal shift assay is a direct andquantitative technology for assaying the effect of one or more moleculeson the thermal stability of a target receptor.

The theory, concepts, and application of the microplate thermal shiftassay, and apparatuses useful for practicing the microplate thermalshift assay are described in U.S. Pat. Nos. 6,020,141; 6,036,920;6,291,191; 6,268,218; 6,232,085; 6,268,158; 6,214,293; 6,291,192; and6,303,322, which are all hereby incorporated by reference in theirentireties. The microplate thermal shift assay discussed in thesereferences can be used to implement the screening methods describedabove.

The microplate thermal shift assay provides a thermodynamic readout ofligand binding affinity. The assay depends upon the fact that eachfunctionally active target molecule is a highly organized structure thatmelts cooperatively at a temperature that is characteristic for eachtarget molecule and representative of its stabilization energy. When amolecule binds to a target molecule, the target molecule is stabilizedby an amount proportional to the ligand binding affinity, thus shiftingthe midpoint temperature to a higher temperature.

There are many advantages to using the thermal shift assay since it doesnot require radioactively labeled compounds, nor fluorescent or otherchromophobic labels to assist in monitoring binding. The assay takesadvantage of thermal unfolding of biomolecules, a general physicalchemical process intrinsic to many, if not all, drug targetbiomolecules. General applicability is an important aspect of thisassay, as it obviates the necessity to invent a new assay every time anew therapeutic receptor protein becomes available.

Further, using the thermal shift assay, owing to the proportionality ofthe T_(m) and the ligand binding affinity, ligand binding affinitiesranging from greater than 10 micromolar to less than 1 nanomolar can bemeasured in a single well experiment. Thus, the thermal shift assay canbe used to quantitatively detect ligand binding affinity to a targetmolecule alone and/or in the presence of a co-regulator.

Further, the thermal shift assay can be used in the identification ofagonists and antagonists on a quantitative basis based upon the changein the T_(m) between the ligand and target molecule and the ligand,target molecule and a co-regulator. The microplate thermal shift assaycan be used to measure multiple ligand binding events on a single targetmolecule as incremental or additive increases of the target molecule'smelting temperature.

The present invention has particular utility in the identification ofligands and the identification of such ligands as agonist or antagonistin nuclear receptors. For example, the present invention may be used todetermine binding affinities for nuclear receptor ligands to predict invivo efficacy, to discriminate ligands as agonist or antagonist topredict biological response, and to identify ligands for orphanreceptors to discover their biological function.

For example, the present invention may be used to identify ligands thatinteract with the ligand binding domain of ER-α and ER-β, the twosubtypes of the estrogen receptor family. These domains contain twoknown binding sites, one for estrogen like compounds and another forco-regulator proteins. The present invention can be used to identifyligands that interact with the estrogen receptor. These ligands producean observed increase in the stability of the receptor which isproportional to the inherent affinity of the ligand.

The ligand binding domain of nuclear receptors, and co-regulatorproteins can be expressed using standard recombinant methods inEscherichia coli. Co-regulator peptides can be synthesized usingstandard methods. The melting temperature of the purified protein ofinterest can be determined by the microplate thermal shift assay in theabsence and in the presence of small molecule ligands.

Molecules are provided that stabilize the target molecule of interest.Such small molecules can be obtained by screening in the microplatethermal shift assay, as referred to above. The number of small moleculesin the screen can range from about one thousand to one million. Thesmall molecules can be natural or synthetic.

Once a set of small molecules have been identified to stabilize theprotein of interest, then these molecules can be screened against apanel of co-regulators, such as proteins or peptide fragments, tomeasure their effect on the thermal stability of the protein. If asynergistic effect is observed, the compounds can be classified asagonist or antagonist. Equilibrium constants are calculated for bothligand and co-regulator and related to biological responses.

For assigning biological function to orphan receptors, the rate limitingstep is the generation of a tool compound. One can screen the receptorof interest against a panel of compounds and identify ligands thatstabilize the receptor of interest by the methods described above. Onceligands are identified, then one can screen against co-regulators todetermine if the identified ligand is an agonist or an antagonist themethods described above.

Cell lines that contain the receptor of interest, as determined by,e.g., Western blot analysis, can be treated with the identified ligand.The ligand treated cell line can then be profiled for gene expressionwith DNA chips and compared against untreated cell lines. If theidentified ligand is an agonist, a number of genes would be expected tobe down-regulated when compared against the untreated cell line. Oncethis information is generated, the biological function of the receptorcan be defined. This information, with the combination ofchemi-informatics and bio-informatics can also assist in developingtherapeutic hypothesis and testing them for the treatment of disease(see, e.g., Giguere, Endocrine Reviews 20:689-725 (1999), incorporatedby reference herein in its entirety.)

Although the ligand binding domain of nuclear receptors, ligands andco-regulators that interact with this domain is described, the inventioncan be extended to the full length protein, in the presence ofadditional regulators and finally in the presence of DNA.

Further, it must be emphasized that the methods and the thermodynamicprinciples for data analysis can be used for any protein-proteininteraction whose affinity is modulated by ligands or allostericregulators. Examples can be and are not limited to GPCR's interactingwith G-proteins to discriminate agonist from antagonist ligands;discriminating compounds that antagonize the association of SH2 domainsto phophorylated forms of protein tyrosine kinases; identifyingcompounds that agonize or antagonize the PKA holoenzyme by affecting theoligomeric state of the enzyme; discriminating compounds that promote orinhibit the association of NF-KB to IKB; or compounds that promote orinhibit the oligomerization of transcription factors.

Also, these studies are not limited for protein-protein interactions butalso can be used for protein-peptide interactions where the peptidesrepresent short linear sequences representing protein domains thatinteract preferentially with the protein of interest.

Having now generally described the invention, the same will become morereadily understood by reference to the following specific examples whichare included herein for purposes of illustration only and are notintended to be limiting unless otherwise specified.

Experimental Results For Nuclear Receptors

The experimental results expected for an agonist response vs. anantagonist response in the presence of a co-activator is shown in FIGS.1 and 2. In the case of an agonist ligand and in the presence ofco-activator protein/peptide the prediction is an increase in thestability of the receptor (FIG. 1), while for an antagonist noadditional stabilization will be observed (FIG. 2).

EXAMPLE 1

Table 1 is a summary of the data obtained for ER-c and ER-β for thestudy of a panel of four known agonist and three known antagonists inthe presence of a co-activator protein SRC-3; in the presence of twoco-activator peptides SRC1—NR2 and SRC3—NR2 derived from the sequence ofthe co-activators SRC-1 and SRC-3; and in the presence of theco-repressor peptide NCoR-1 derived from the co-repressor NCoR-1.

The concentration of ER-α and ER-β in all of the experiments was 8 μM,the ligand concentration was 20 μM, SRC-3 was 11 μM, and theco-regulator peptides SRC1—NR2, SRC3—NR2, and NCoR-1 was at 100 XM. Theexperiments were performed in 25 mM phosphate pH 8.0, 200 mM NaCl, 10%glycerol and in the presence of 25 μM dapoxyl sulfonamide dye (availablefrom Molecular Probes, Inc., Eugene, Oreg.).

A 2 μL ligand solution at 2 times the final concentration was dispensedwith a micropipette into a 384 well black wall Greiner plate. Then, 2 μLof the protein dye solution was dispensed on top of the ligand solutionin the 384 well plate. The plates were spun to ensure mixing of theprotein-dye and ligand solutions followed by layering of 1 μL ofsilicone oil to prevent evaporation during heating of the samples. Datawere collected on a Thermofluor apparatus (see U.S. Pat. Nos. 6,020,141;6,036,920; 6,291,191; 6,268,218; 6,232,085; 6,268,158; 6,214,293;6,291,192; and 6,303,322) and analyzed using non-linear least squaresfitting software. The results listed below are the average of fourexperiments. The values for the co-regulators represent a change inT_(m) stabilization from the receptor-ligand ΔT_(m) values. TABLE 1Observed ΔTm stabilization of estrogen receptors in the presence ofligands and coregulators. — SRC-3 SRC-1 NR2 SRC-3 NR2 NCoR1 ER-α 0.0 0.50.8 0.9 0.0 Estradiol 13.4 3.8 4.9 4.3 −0.2 Estrone 9.1 2.0 3.0 2.3 −0.317-α-ethylen-E2 14.8 3.7 4.5 3.9 −0.2 2-methoxy-E2 1.8 4.1 5.5 4.3 −0.3tamoxifen 9.4 0.5 −0.5 0.0 0.1 4-OH-tamoxifen 16.7 0.2 0.2 0.7 0.1ICI-182780 13.8 0.0 0.2 0.2 −0.6 ER-β 0.0 0.4 0.7 0.9 −0.4 Estradiol16.4 1.1 3.4 3.5 0.0 Estrone 8.9 0.8 3.8 3.7 −0.3 17-a-ethylene 15.0 1.62.6 2.6 −0.1 2-methoxy-E2 1.5 2.2 4.4 4.4 −0.7 tamoxifen 6.1 0.2 0.2 0.40.4 4-OH-tamoxifen 14.7 0.4 0.2 0.8 0.3 ICI-182780 13.6 0.6 0.4 0.6 0.3

From the above results, from counter-screening in the presence ofco-activator protein/peptide in the presence of the estrogen-likecompounds, an additional stabilization was observed for both receptors.Thus, these compounds act like agonists in agreement with literature.The tamoxifen and ICI compound are known antagonists and they have noability to recruit co-activators. This is also in agreement with theliterature.

Also, the co-activator SRC-3 is preferentially recruited by ER-(c vs.ER-β. Therefore, the prediction is that these estrogen like compoundshave a higher biological response in cell lines that contain ER-α vs.ER-β in the presence of SRC-3.

Further, the estrogen receptor does not have ability to recruitco-repressor peptide, therefore from a biological point of view theprediction is that gene repression will occur in ligand dependentfashion.

EXAMPLE 2

ER-α was screened against a panel of steroid-like ligands to verify theability of the methods of the present invention to determine ligands,and the function (see U.S. Patent Publication No. U.S. 2001/0003648 A1),of ER-(X if this receptor was classified as an orphan. Ligands that areknown to interact with ER-α are identified as producing an increase inthe stability of the receptor (compounds that are underlined versusthose which are not underlined).

The concentration of ER-α in all of the experiments was 8 μM and theligand concentration was 20 μM. The experiments were performed in 25 mMphosphate pH 8.0, 200 nM NaCl, 10% glycerol and in the presence of 25 μMdapoxyl sulfonamide dye (available from Molecular Probes, Inc., Eugene,Oreg.).

A 2 μL ligand solution at 2 times the final concentration was dispensedwith a micropipette into a 384 well black wall Greiner plate. Then, 2 μLof the protein dye solution was dispensed on top of the ligand solutionin the 384 well plate. The plates were spun to ensure mixing of theprotein-dye and ligand solutions followed by layering of 1, of siliconeoil to prevent evaporation during heating of the samples. Data werecollected on a Thermofluor apparatus (see U.S. Pat. Nos. 6,020,141;6,036,920; 6,291,191; 6,268,218; 6,232,085; 6,268,158; 6,214,293;6,291,192; and 6,303,322) and analyzed using non-linear least-squaresfitting software. The results listed below are the average of fourexperiments. TABLE 2 Summary of data for ER-α in the presence of a panelof steroid ligands Steroid Ligand ΔTm Receptor target 4-androstene −0.23androgen receptor androsterone 0.23 androgen receptor corticosterone−0.27 glucocorticoid receptor cortisone 0.01 glucocorticoid receptorβ-estradiol 15.19 estrogen receptor estrone 9.91 estrogen receptor17-α-ethyleneestradiol 18.72 estrogen receptor 17-α-hydroxyprogesterone−0.21 progesterone receptor 2-methoxyestradiol 5.98 estrogen receptorquabain −0.21 progesterone receptor progesterone −0.19 progesteronereceptor 4-hydroxytamoxifen 19.99 estrogen receptor

If ER-α was an orphan receptor, the data would had been interpreted thatthis receptor is a member of the estrogen receptor family. If theidentified ligands that bind to the receptor had been screened against apanel of co-regulators, as in Example 1, β-estradiol, estrone,17-α-ethyleneestradiol, and 2-methoxyestradiol are agonists for thisreceptor, while 4-hydroxytamoxifen is an antagonist. This data setdemonstrates the utility of the microplate thermal shift assay for theidentification of ligands for orphan receptors.

EXAMPLE 3

Examples of other protein-protein interactions that may be analyzedusing the present invention are illustrated in Table 2. TABLE 2Embodiment examples Protein Protein of Partner (co- Ligand Interestregulator) Phenotype Related Biological Activity GPCR Gsα AgonistIncrease cAMP or stimulate regulation of Ca²⁺ channels GPCR Giα AgonistDecrease cAMP GPCR Goα Agonist Stimulate regulation of Ca²⁺ channelsGPCR Gtα Agonist Increase cGMP and phosphodiesterase activity GPCR GqαAgonist Increase phospholipase Cβ activity GPCR Gsα Antagonist No effecton basal activity, or decrease cAMP, or inhibition of Ca²⁺ channelstimulation GPCR Giα Antagonist No effect on basal activity, or increasecAMP GPCR Goα Antagonist No effect on basal activity, or inhibition ofCa²⁺ channel stimulation GPCR Gtα Antagonist No effect on basalactivity, or decrease cGMP and phosphodiesterase activity GPCR GqαAntagonist No effect on basal activity, or decrease phospholipase Cβactivity Src SH2 Antagonist Inhibition of osteoclast mediated resorptionof bone Src SH2 Agonist Stimulation of osteoclast mediated resorption ofbone Jac SOCS Agonist Gene transcription Jac SOCS Antagonist Generepression NF-κB IκB Antagonist Gene transcription NF-κB IκB AgonistGene repression

Different embodiments of this invention can include and are not limitedto the examples above. The general nature of the examples contain theprotein of interest, the interacting protein or peptide partner(co-regulator, e.g., a co-activator or co-repressor), and the ligandthat can enhance (an agonist) or inhibit (an antagonist) theinteraction.

While the foregoing invention has been described in some detail for thepurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1-7. (canceled)
 8. A method of identifying an agonist or an antagonist of a co-regulatordependent target molecule comprising providing a set of molecules that shift the thermal unfolding curve of the target molecule and screening one or more of said molecules of said set for their ability to further shift the thermal unfolding curve of the target molecule in the presence of one or more coregulators; wherein a further shift in the thermal unfolding curve of the target molecule in the presence of a molecule of said set and a co-regulator of said one or more co-regulators indicates whether the molecule is an agonist or an antagonist of the target molecule when in the presence of said co-regulator.
 9. The method of claim 8, wherein providing the set of molecules that shift the thermal unfolding curve of the target molecule comprises screening one or more of a multiplicity of different molecules for their ability to shift the thermal unfolding curve of the target molecule.
 10. The method of claim 9, wherein the screening of said one or molecules of said set of molecules for their ability to further shift the thermal unfolding curve of the target molecule comprises: (a) contacting said target molecule and one or more molecules of said set with one or more of said co-regulators in each of a multiplicity of containers; (b) heating said multiplicity of containers; (c) measuring in each of said containers a physical change associated with the thermal unfolding of said target molecule resulting from said heating; (d) generating a thermal unfolding curve for said target molecule as a function of temperature for each of said containers; (e) comparing each of said thermal unfolding curves in step (d) to (1) each of the other thermal unfolding curves and/or to (2) the thermal unfolding curve for said target molecule in the absence of (i) any of said molecules of said set and/or (ii) said co-regulators, and (f) determining whether any of said molecules of said set further shift the thermal unfolding curve of said target molecule.
 11. The method of claim 10, wherein the screening of said one or more of a multiplicity of different molecules for their ability to shift the thermal unfolding curve of the target molecule comprises: (a) contacting said target molecule with one or more of said multiplicity of different molecules in each of a multiplicity of containers; (b) heating said multiplicity of containers; (c) measuring in each of said containers a physical change associated with the thermal unfolding of said target molecule resulting from said heating; (d) generating a thermal unfolding curve for said target molecules as a function of temperature for each of said containers; (e) comparing each of said thermal unfolding curves in step (d) to (1) each of the other thermal unfolding curves and/or to (2) the thermal unfolding curve for said target molecule in the absence of any of said multiplicity of different molecules; and determining whether and of said multiplicity of different molecules shift the thermal unfolding curve of said target molecule.
 12. The method of claim 8, wherein said one or more co-regulators includes a co-activator and/or a co-repressor.
 13. The method of claim 12, wherein one or more molecules of the set further shift the thermal unfolding curve of the target molecule in the presence of a co-activator, thereby identifying an agonist of the target molecule when in the presence of the co-activator.
 14. The method of claim 12, wherein the one or more molecules of the set further shift the thermal unfolding curve of the target molecule in the presence of a co-repressor, thereby identifying an antagonist of the target molecule when in the presence of the co-repressor. 15-18. (canceled)
 19. A method of identifying an antagonist of a co-regulator-dependent target molecule comprising providing a set of molecules that shift the thermal unfolding curve of the target molecule and screening one or more of said molecules of said set for their ability to further shift the thermal unfolding curve of the target molecule in the presence of one or more co-activators; wherein no further shift in the thermal unfolding curve in the presence of a molecule of said set and a co-activator of said one or more-co-activators indicates that the molecule of said set is an antagonist of the target molecule when in the presence of said co-activator.
 20. The method of claim 19, wherein providing the set of molecules that shift the thermal unfolding curve of the target molecule comprises screening one or more of a multiplicity of different molecules for their ability to shift the thermal unfolding curve of the target molecule.
 21. The method of claim 20, wherein the screening of said one or molecules of said set of molecules for their ability to further shift the thermal unfolding curve of the target molecule comprises: (a) contacting said target molecule and one or more molecules of said set with one or more of said co-activators in each of a multiplicity of containers; (b) heating said multiplicity of containers; (c) measuring in each of said containers a physical change associated with the thermal unfolding of said target molecule resulting from said heating; (d) generating a thermal unfolding curve for said target molecule as a function of temperature for each of said containers; (e) comparing each of said thermal unfolding curves in step (d) to (1) each of the other thermal unfolding curves and/or to (2) the thermal unfolding curve for said target molecule in the absence of (i) any of said molecules of said set and/or (ii) said co-activators; and (f) determining whether any of said molecules of the set further shift the thermal unfolding curve of said target molecule.
 22. The method of claim 21, wherein the screening of said one or more of a multiplicity of different molecules for their ability to shift the thermal unfolding curve of the target molecule comprises: (a) contacting said target molecule with one or more of said multiplicity of different molecules in each of a multiplicity of containers; (b) heating said multiplicity of containers; (c) measuring in each of said containers a physical change associated with the thermal unfolding of said target molecule resulting from said heating; (d) generating a thermal unfolding curve for said target molecule as a function of temperature for each of said containers; (e) comparing each of said thermal unfolding curves in step (d) to (1) each of the other thermal unfolding curves and/or to (2) the thermal unfolding curve for said target molecule in the absence of any of said multiplicity of different molecules; and (f) determining whether any of said multiplicity of different molecules shift the thermal unfolding curve of said target molecule. 23-26. (canceled)
 27. A method of identifying an agonist of a co-regulator-dependent target molecule comprising providing a set of molecules that shift the thermal unfolding curve of the target molecule and screening one or more of said molecules of said set for their ability to further shift the thermal unfolding curve of the target molecule in the presence of one or more co-repressors; wherein no further shift in the thermal unfolding curve in the presence of a molecule of said set and a co-repressor of said one of more-co-repressors indicates that the molecule of said set is an agonist of the target molecule when in the presence of said co-repressor.
 28. The method of claim 27, wherein providing the set of molecules that shift the thermal unfolding curve of the target molecule comprises screening one or more of a multiplicity of different molecules for their ability to shift the thermal unfolding curve of the target molecule.
 29. The method of claim 28, wherein the screening of said one or molecules of said set of molecules for their ability to further shift the thermal unfolding curve of the target molecule comprises: (a) contacting said target molecule and one or more molecules of said set with one or more of said co-repressors in each of a multiplicity of containers; (b) heating said multiplicity of containers; (c) measuring in each of said containers a physical change associated with the thermal unfolding of said target molecule resulting from said heating; (d) generating a thermal unfolding curve for said target molecule as a function of temperature for each of said containers; (e) comparing each of said thermal unfolding curves in step (d) to (1) each of the other thermal unfolding curves and/or to (2) the thermal unfolding curve for said target molecule in the absence of (i) any of said molecules of said set and/or (ii) said co-repressors; and (f) determining whether any of said molecules of the set further shift the thermal unfolding curve of said target molecule.
 30. The method of claim 29, wherein the screening of said one or more of a multiplicity of different molecules for their ability to shift the thermal unfolding curve of the target molecule comprising: (a) contacting said target molecule with one or more of said multiplicity of different molecules in each of a multiplicity of containers; (b) heating said multiplicity of containers; (c) measuring in each of said containers a physical change associated with the thermal unfolding of said target molecule resulting from said heating; (d) generating a thermal unfolding curve for said target molecule as a function of temperature for each of said containers; (e) comparing each of said thermal unfolding curves in step (d) to (1) each of the other thermal unfolding curves and/or to (2) the thermal unfolding curve for said target molecule in the absence of any of said multiplicity of different molecules; and (f) determining whether any of said multiplicity of different molecules shift the thermal unfolding curve of said target molecule. 31-34. (canceled)
 35. The method of claim 8, wherein the target molecule is a nuclear receptor.
 36. The method of claim 8, wherein the target molecule is a G-protein coupled receptor.
 37. The method of claim 8, wherein the target molecule is ER-α.
 38. The method of claim 8, wherein the target molecule is ER-β.
 39. The method of claim 8, wherein the target molecule is a tyrosine kinase.
 40. The method of claim 8, wherein the target molecule is a NF-κB. 